This invention relates to the inhibition of blood coagulation, especially during organ rejection.
The surgical technique of organ transplantation has now been successfully practised for several decades and, because of its success, the procedure has become widespread and, arguably, routine. However, the supply of suitable transplant organs is not able to match ever-rising demands.
Because of the shortage of suitable human (ie. allogeneic) organs, the possibility of using animal (ie. xenogeneic) organs in human transplant operations (xe2x80x9cxenograftingxe2x80x9d or xe2x80x9cxenotransplantationxe2x80x9d) has been receiving increased attention in recent years (eg. Nature 1997; 385:285). Porcine donor organs are thought to be suitable candidates because pigs are anatomically and physiologically similar to humans and are in abundant supply.
Xenografting is currently hindered, however, by the severe and well-documented problems of rejection. This process can be divided into distinct stages, the first of which occurs within minutes of transplantation. This is known as the hyperacute response and is caused by existing antibodies in the recipient which recognise and react with foreign antigens on the endothelial cells (ECs) of the xenograft. This recognition triggers the complement cascade which in turn leads to lysis and death of ECs of the transplant.
This initial hyperacute rejection is then reinforced by the delayed vascular response (also known as acute vascular rejection or delayed xenograft rejection). The lysis and death of ECs during the hyperacute response is accompanied by oedema and the exposure of adventitial cells, which constitutively express tissue factor (TF) on their surface. Tissue factor is thought to be pivotal in the initiation of the in vivo coagulation cascade, and its exposure to plasma triggers the clotting reactions. Thrombin and TNF-xcex1 become localised around the damaged tissue and this induces further synthesis and expression of TF by ECs.
The environment around resting ECs does not favour coagulation. Several natural coagulation inhibitors are associated with the extracellular proteoglycans of ECs, such as tissue factor pathway inhibitor, antithrombin III, and thrombomodulin. The recognition of the foreign tissue by xenoreactive natural antibodies (XNAs), however, causes the loss of these molecules.
Together with the exposure and induction of tissue factor, the anticoagulant environment around ECs thus becomes pro-coagulant.
The vascularised regions of the xenograft thus become sites of blood clots, a characteristic of damaged tissue. Blood flow is impaired and the transplanted organ becomes ischaemic. A fuller account of delayed vascular rejection can be found in Bach et al. (1996).
The use of xenogeneic organs in transplants is therefore hindered by an initial hyperacute rejection followed by a prolonged vascular rejection, possibly followed by T-cell mediated rejection. Inhibition of the mechanisms responsible for these rejections could facilitate the use of xenografts.
The simple administration of suitable inhibitors, however, is not a particularly suitable approach. Completely inhibiting complement in a recipient animal is tantamount to immunosuppression, leaving the subject prone to opportunistic infections. Similarly, inhibiting the coagulation cascade in a recipient will leave the animal susceptible to uncontrolled post-operative bleeding. Therefore the inhibitors should desirably be localised in the recipient to the site of the xenograft.
The prevention of hyperacute rejection is the subject of European patent 0495852 (Imutran). To make tissues more suitable for xenografting this patent teaches that they should be associated with homologous complement restriction factors, which prevent the complete activation of complement in the xenogeneic organ recipient.
This approach has been developed and applied in order to produce transgenic animals with organs designed to survive hyperacute rejection (Squinto, 1996). Transgenic mice expressing human CD59, a complement regulator, on cardiac ECs have been produced (Diamond, 1995). The human CD59 retained biological activity and complement was inhibited when transgenic hearts were perfused with human plasma.
Transgenic pigs expressing human DAF and/or CD59 have been reported (McCurry, 1996). Cardiac rejection took twice as long to occur with the transgenic xenografts than with controls.
Inhibiting delayed vascular rejection has not received the same attention, although inhibitors of the coagulation cascade are well known in the art and many have been well characterised.
For instance, tissue factor pathway inhibitor (TFPI) is known to inhibit the function of the active complex which is normally formed between tissue factor, factor VIIa, and factor Xa. TFPI is a 276 residue soluble polypeptide whose positively charged C-terminus binds to heparin sulphate in the proteoglycan layer of ECs. It has been notionally divided into three xe2x80x9cKunitzxe2x80x9d domains: Kunitz domain I is responsible for binding tissue factor and factor VIIa; domain II binds factor Xa; but the functions of domain III are less clear (Hamamoto, 1993).
Tick anticoagulant peptide (TAP) is a specific and potent inhibitor of factor Xa. This 60 amino acid polypeptide has been purified from the soft tick Ornithodoros moubata. 
Many snake venoms also contain anticoagulant polypeptides. For instance, a 231 amino acid protein C activator has been purified from the venom of the snake Agkistrodon contortrix contortrix (McMullen, 1989; Kisiel, 1987).
Hirudin is the anticoagulant protein utilised by the leech Hirudo medicinalis when extracting blood from its victim. It is highly potent and binds to thrombin at a 1:1 ratio with a dissociation constant in the femtomolar range. The active site of thrombin is masked in the stable complex and so the hirudin prevents fibrinogen breakdown, thus inhibiting clot formation.
One possible approach for localising anticoagulants to the site of rejection is to link hirudin to antibodies against E-selectin, which is expressed on the surface of ECs during cell activation. This approach has been shown to be effective in inhibiting clot formation in vitro (Kiely, 1995). Other possible strategies were recently reviewed by Bach et al. (1996).
P-selectin (also known as CD62) is also expressed on the surface of ECs during cell activation. During synthesis it is targeted to secretory storage granules in platelets and endothelial cells by sequences residing in its cytoplasmic domain (Disdier, 1992). In response to cell agonists, such as thrombin, the granules are rapidly redistributed and P-selectin is expressed on the cell surface (Green, 1994).
It is an object of the present invention to provide membrane-bound anticoagulant proteins. These proteins are suitable for inhibiting the clotting cascade at the surface of ECs, thus inhibiting in vivo mechanisms responsible for organ rejection.
It is a further object to provide regulated expression of such molecules on the surface of ECs such that coagulation inhibition occurs locally during conditions of organ rejection. The rejection might be xenogeneic or allogeneic.
It is yet a further object of the invention to provide biological tissue suitable for transplantation, particularly for xenotransplantation.
According to a first aspect of the present invention there is provided a protein comprising a region with anticoagulant activity and a region which can anchor said protein to a cell membrane. Preferably this is a chimeric protein, that is to say the anchor region and anticoagulant region are derived from different proteins.
The anticoagulant region can comprise the sequence of any anticoagulant polypeptide. Examples of such anticoagulant polypeptides include heparin, TAPs, antithrombin, hirudins, and TFPIs, along with their functional derivatives, such as fragments and derivatives which retain anticoagulant activity. Anticoagulant derivatives of thrombin, normally a procoagulant, have also been reported (Dang, 1997).
Preferably the anticoagulant region comprises the sequence of a hirudin. Hirudins include hirudin, hirudin derivatives, analogs (xe2x80x9chirulogsxe2x80x9d), and variants (eg. hirudisins). For instance, it has been reported that sulphation at Tyr-64 increases the anticoagulant activity of hirudin, and that hirudisin-2 is a more potent inhibitor of thrombin activity than hirudin itself (eg. Knapp, 1992; Skern, 1990).
As an alternative, the anticoagulant region might comprise the sequence of a tissue factor pathway inhibitor (TFPI). TFPIs include TFPI itself and derivatives or analogs thereof which retain inhibitory activity. Preferably the TFPI sequence comprises Kunitz domains I and II of TFPI itself.
As a further alternative, the anticoagulant region might comprise the sequence of a tick anticoagulant peptide (TAP). TAPs include TAP itself and derivatives or analogs thereof which retain inhibitory activity. For instance, the potency of FXa inhibition by TAP has been enhanced by site-directed mutagenesis (eg. Mao, 1995).
Further alternative anticoagulant regions could, for instance, comprise the sequence of a protein C activator, such as those isolated from snake venom (eg. McMullen, 1989; Kisiel, 1987), or the sequence of anticoagulants isolated from snake venoms which act other than via protein C activation, or their derivatives or analogs which retain anticoagulant activity.
The anchor region can be any entity which can attach the protein to a cell membrane. Suitable examples include transmembrane sequences from membrane proteins and GPI anchors. Preferably the anchor region is a sequence capable of attaching the protein to a lipid bilayer, such as the transmembrane regions of the HLA class I or CD4 proteins. It may also be desirable for the protein to comprise the cytoplasmic domain which is usually associated with said transmembrane regions, such as the CD4 cytoplasmic domain, and/or the extracellular domains immediately juxtaposed with the cell membrane, such as CD4 domains 3 and 4. Alternatively the anchor region might be a sequence conferring on the protein the ability to associate extracellularly with a membrane protein without the protein itself being inserted into the cell membrane.
According to a second aspect of the invention, there is provided a protein according to the first aspect further comprising a targeting sequence which prevents the protein from being constitutively expressed at the cell surface.
Preferably the targeting sequence is a polypeptide sequence which can target a nascent polypeptide to a secretory granule, and more preferably the secretory granule is one which does not fuse with the cell""s plasma membrane until the cell is suitably stimulated. For example, Weibel-Palade bodies do not fuse with the plasma membrane until the endothelial cell surface is stimulated by a secretagogue, such as thrombin or fibrin (Wagner, 1993). Preferably the secretory granule fuses with the plasma membrane during EC activation which occurs during organ rejection.
Thus the targeting sequence is preferably one which targets a nascent polypeptide to a Weibel-Palade body, such as the relevant sequence from P-selectin. Most preferably the protein according to the second aspect of the invention comprises an anticoagulant sequence and the transmembrane and cytoplasmic domains of P-selectin. The domains from P-selectin thus provide both the anchor sequence and the targeting sequence.
According to a third aspect of the invention, there is provided a polynucleotide encoding a protein according to the present invention. Preferably the polynucleotide is DNA.
Preferably the polynucleotide comprises sequences suitable for the regulation of expression of protein according to the invention. This expression can preferably be controlled, such as cell-specific control, inducible control, or temporal control. For instance, expression might be specific for ECs, or might be regulated in response to cell activation.
According to a fourth aspect of the invention, there is provided a vector comprising a polynucleotide according to the third aspect.
The term xe2x80x9cvectorxe2x80x9d signifies a molecule which is capable of transferring a polynucleotide to a host cell. Preferably the vector is a DNA vector and, more preferably, is capable of ex pressing RNA encoding a protein according to the invention. Numerous suitable vectors are known in the art.
Preferably the vector is suitable for the production of a transgenic animal. Vectors suitable for the generation of transgenic pigs, for example, are described in Heckl-xc3x96streicher (1995), McCurry (1996), White (1995), Yannoutsos (1995), and Langford (1996). Minigene vectors suitable for the generation of transgenic mice are described in Diamond (1995).
According to a fifth aspect of the invention, there is provided a delivery system comprising a molecule of the first, second , third, or fourth aspects and means to deliver said molecule to a target cell.
Certain vectors according to the fourth aspect may also function as suitable delivery systems. Likewise, certain delivery systems according to this fifth aspect may also inherently be vectors, but this is not always the case. For instance, a viral vector can also function as a delivery system, whereas a liposomal delivery system is not a vector.
The delivery system may be viral or non-viral. Non-viral systems, such as liposomes, avoid some of the difficulties associated with virus-based systems, such as the expense of scaled production, poor persistence of expression, and concerns about safety. Preferably the delivery system is suitable for use in gene therapy. Numerous appropriate delivery systems are known in the art.
Preferably, the delivery system will be targeted so that molecules according to the present invention are taken up by cells suitable for transplantation, or cells which have been transplanted. More preferably the delivery system will be specific for these cells. For example, the delivery system may be targeted to a specific organ, such as the heart or the kidney, or to a specific cell type, such as endothelial cells.
To achieve this the delivery system may, for example, be a receptor-mediated delivery system, being targeted to receptors found on target cells. For example, the delivery system may be targeted to receptors found on heart cells, preferably to receptors found exclusively on heart cells, or it may be targeted to receptors found on endothelial cells, preferably to receptors found exclusively on endothelial cells, or to receptors found on activated endothelial cells, such as E-selectin or P-selectin.
The delivery system is preferably suitable for the generation of transgenic animals. For example, the delivery system may be targeted to a gamete, a zygote, or an embryonic stem cell.
According to a sixth aspect of the invention, there is provided a method of transfecting a cell with a vector according to the invention. This may involve the use of a delivery system according to the invention.
The cell type is not restricted and may be prokaryotic or eukaryotic. Transfection can occur in vivo or ex vivo.
Where the cell is for use in transplantation, the cell is preferably eukaryotic, more preferably an endothelial cell. The stable transfection of porcine endothelial cells, for example, is described in Heckl-xc3x96streicher (1995).
Preferably, the cell is suitable for the generation of a transgenic animal. More preferably, the cell is a gamete, a zygote, or an embryonic stem cell. The transfection of murine ova by microinjection to generate transgenic mice, for example, is described in Diamond (1995), and the microinjection of porcine zygotes, for instance, to generate transgenic pigs is described in Yannoutsos (1995), Langford (1996), and White (1995).
According to a seventh aspect of the invention, there is provided a cell transfected according to the sixth aspect.
To increase the efficacy of inhibition of the coagulation cascade, the cell is preferably able to express two or more different proteins according to the invention, each of which inhibits the coagulation cascade at a different stage. For example, the anticoagulant region in one protein might comprise a TFPI, whilst in the other it comprises a hirudin.
According to an eighth aspect of the invention, there is provided biological tissue comprising a cell according to the invention. The term xe2x80x9cbiological tissuexe2x80x9d as used herein includes collections of cells, tissues, and organs. Accordingly the definition includes, for example, fibroblasts, a cornea, nervous tissue, a heart, a liver, or a kidney.
According to a ninth aspect of the invention, there is provided an animal comprising a cell and/or biological tissue according to the invention. Preferably the animal is suitable for the production of organs for transplantation into humans. Preferably the animal is a mammal, and more preferably it is a transgenic pig or a transgenic sheep.
The animal might be treated whilst alive such that it comprises transgenic biological tissue (i.e. treated by gene therapy). Preferably, a live animal is transfected with a vector according to the invention in order to produce a transgenic animal. For example, a vector according to the invention could be specifically delivered to endothelial cells in a pig to produce transgenic organs suitable for xenotransplantation.
Alternatively, the animal might be born as a transgenic animal. Various suitable approaches for generating such transgenic animals are known in the art (eg. Bradley and Liu, 1996; Clarke, 1996; Wheeler, 1994). For example, direct manipulation of the zygote or early embryo, by microinjection of DNA for instance, is well known, as is the in vitro manipulation of pluripotent cells such as embryonic stem cells. Retroviral infection of early embryos has proved successful in a range of species, and adenoviral infection of zona-free eggs has been reported. Transgenesis and cloning of sheep by nuclear transfer has also been described (eg WO97/07668).
According to a tenth aspect of the invention, there is provided a method of rendering biological tissue suitable for transplantation, comprising expressing one or more proteins according to the present invention in said biological tissue, preferably in its endothelial cells. The biological tissue may be so rendered either in vivo or ex vivo. For example, an animal organ may be in vivo transfected with a vector according to the invention, or an organ could be transfected ex vivo before transplantation or in vivo after transplantation.
According to an eleventh aspect of the invention, there is provided a method of transplantation comprising transplanting biological tissue according to the invention from a donor animal into a recipient animal. Preferably the method is for xenotransplantation and the donor biological tissue is xenogeneic with respect to the recipient animal.